Ca+ activity maps of astrocytes tagged by axoastrocytic AAV transfer

Astrocytes exhibit localized Ca2+ microdomain (MD) activity thought to be actively involved in information processing in the brain. However, functional organization of Ca2+ MDs in space and time in relationship to behavior and neuronal activity is poorly understood. Here, we first show that adeno-associated virus (AAV) particles transfer anterogradely from axons to astrocytes. Then, we use this axoastrocytic AAV transfer to express genetically encoded Ca2+ indicators at high-contrast circuit specifically. In combination with two-photon microscopy and unbiased, event-based analysis, we investigated cortical astrocytes embedded in the vibrissal thalamocortical circuit. We found a wide range of Ca2+ MD signals, some of which were ultrafast (≤300 ms). Frequency and size of signals were extensively increased by locomotion but only subtly with sensory stimulation. The overlay of these signals resulted in behavior-dependent maps with characteristic Ca2+ activity hotspots, maybe representing memory engrams. These functional subdomains are stable over days, suggesting subcellular specialization.


The PDF file includes:
Figs. S1 to S10 Table S1 Legends for movies S1 to S3

Other Supplementary Material for this manuscript includes the following:
Movies S1 to S3 í Supplementary Figures   Fig. S1. Barrel cortex astrocytes and neurons can be labelled with a single AAV injection in VPM.

(A)
AAV1-hSyn-TurboRFP and AAV1-CAG-GCaMP6f injected in VPM label neurons and astrocytes in BX with GCaMP6f. Thalamocortical projections are double labeled with GCaMP6f and TurboRFP. This indicates that intersectional approaches or Cre-recombinase are not required for AAV mediated anterograde transduction of BX astrocytes and neurons. Note: AAV1-hSyn-TurboRFP will also transfer to BX neurons, however the expression level under the hSyn promoter is too low to be visible here.

(B)
AAV1-CMV-Cre and AAV1-CAG-Flex-eGFP injected in VPM labels neurons and astrocytes in BX with eGFP. This indicates that intersectional approaches and/or a specific fluorescent protein (i.e., GCaMP6f) are not required for AAV mediated anterograde transduction of astrocytes and neurons.
Confocal images were taken from fixed brain slices of mice sacrificed two weeks after injections.

(B)
Percentage of GCaMP6f labelled cells in BX that are S100β positive (black bar) and S100β negative (gray bar) in mice injected as in (A) (n = 32 cells, 12 slices, 3 mice).

(D)
Percentage of GCaMP6f labelled cells in BX that are NeuN positive (black bar) and NeuN negative (gray bar) in mice injected as in (C) (n = 70 cells, 9 slices, 3 mice).

(D)
Neurons (green) and thalamocortical axons (red) distributed in the six cortical layers in BX. Scale bar 50 μm.

(F)
Mean density ± CI of neurons in L1, L2/3, L4 and L5/6 of the BX. Probability density f(X) of event amplitudes (ΔF/F) during rest (gray dots) and run (black dots) states, fit by skewed distribution functions, corresponding to rest (blue line) and run (red line, black dots) states.

(B)
Probability density (f(X)) of event durations (s) during rest (gray dots) and run (black dots) states, fit by skewed distribution functions, corresponding to rest (blue line) and run (red line) states.
Number of recordings = 9, number of mice = 3, number of astrocytes = 6. Total imaging time and ratio of total rest and run time for the 9 astrocyte recordings analyzed.

Fig. S6. Simultaneous imaging of axonal and astrocytic Ca 2+ MD activity after axoastrocytic AAV transfer. (A)
AAV injection strategy to study axon -astrocyte interactions in BX with two-photon microscopy.

(C)
Mean number of axonal Ca 2+ events/second during rest to run transitions (8613 events, 174 intervals).

(D)
Mean number of axonal Ca 2+ events/second during run to rest transitions (2274 events, 52 intervals)

(E)
Mean number of axonal Ca 2+ events/second 2s before and 4s after the onset of vibrissa stimulation (orange line) (28232 events, 756 intervals).

(G)
Probability distribution of vibrissa stimuli during vibrissa stimulation trials (756 intervals). Axonal activity was restricted to axons outside the astrocyte territory. Data represented as ± 95% confidence interval.
Mean ± CI (black line). One-way repeated measures ANOVA, p < 0.05 *, p > 0.05 nonsignificant (n.s.). Data was normalized to the mean Ca 2+ activity (respective event characteristic) of the same recording during rest state of the animal. Cutoff frequency = 3Hz.

Fig. S8. Ca 2+ hotspots are not randomly distributed (A)
Natural, astrocyte activity heatmap (all states) during day 0 (top left) and day 1 (bottom left) compared to their respective simulated heatmaps generated by random distribution of events (right). All heatmaps are saturated to their 70% maximum pixel value. Simulated heatmaps are normalized to the same range of pixel values as their respective natural heatmap.

(B)
Probability mass functions of normalized pixel intensity values in natural (Real activity heatmap, top) and simulated (Random distribution, bottom) heatmaps. Mean frequency of pixel intensity values (normalized to 1) of 9 astrocyte recordings (mean ± CI). Pixels with higher intensity values than expected of random distributions (within red box) are associated with hotspot activity.

Fig. S9. Heatmap correlations (A)
Pearson's correlation coefficient between the full activity heatmaps of 70-min-long astrocyte recordings (n = 5, 70 min each, colored lines) and their respective subsequences of different duration. Black line = mean ± CI.

(B)
Pearson's correlation coefficient between rest state subsequences (3 per astrocyte, 5 astrocytes) vs: their respective simulated heatmaps (gray), other subsequences of same day recordings during rest state (red), run state heatmaps of same day recordings (black), and subsequences derived from the same astrocyte, recorded a day later, corresponding to rest state (blue). Data represented as mean ± CI. Compared using one-way ANOVA with Tukey's HSD, p < 0.05 *, p < 0.001 ***
AAV1-hSyn-TurboRFP is expected to anterogradely transfer to astrocytes and neurons. Due to the hSyn promoter, TurboRFP is expected to be expressed in BX neurons. However, the expression level was not high enough to be detected.